Exposure to rosiglitazone, a PPAR-γ agonist, in late gestation reduces the abundance of factors regulating cardiac metabolism and cardiomyocyte size in the sheep fetus.

It is unknown whether cardiomyocyte hypertrophy and the transition to fatty acid oxidation as the main source of energy after birth is dependent on the maturation of the cardiomyocytes' metabolic system, or on the limitation of substrate availability before birth. This study aimed to investigate whether intrafetal administration of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, during late gestation can stimulate the expression of factors regulating cardiac growth and metabolism in preparation for birth, and the consequences of cardiac contractility in the fetal sheep at ∼140 days gestation. The mRNA expression and protein abundance of key factors regulating growth and metabolism were quantified using quantitative RT-PCR and Western blot analysis, respectively. Cardiac contractility was determined by measuring the Ca(2+) sensitivity and maximum Ca(2+)-activated force of skinned cardiomyocyte bundles. Rosiglitazone-treated fetuses had a lower cardiac abundance of insulin-signaling molecules, including insulin receptor-ß, insulin receptor substrate-1 (IRS-1), phospho-IRS-1 (Tyr-895), phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, PI3K catalytic subunit p110α, phospho-3-phosphoinositide-dependent protein kinase 1 (Ser-241), protein kinase B (Akt-1), phospho-Akt (Ser-273), PKCζ, phospho-PKCζ(Thr-410), Akt substrate 160 kDa (AS160), phospho-AS160 (Thr-642), and glucose transporter type-4. Additionally, cardiac abundance of regulators of fatty acid ß-oxidation, including adiponectin receptor 1, AMPKα, phospho-AMPKα (Thr-172), phospho-acetyl CoA carboxylase (Ser-79), carnitine palmitoyltransferase-1, and PGC-1α was lower in the rosiglitazone-treated group. Rosiglitazone administration also resulted in a decrease in cardiomyocyte size. Rosiglitazone administration in the late-gestation sheep fetus resulted in a decreased abundance of factors regulating cardiac glucose uptake, fatty acid ß-oxidation, and cardiomyocyte size. These findings suggest that activation of PPAR-γ using rosiglitazone does not promote the maturation of cardiomyocytes; rather, it may decrease cardiac metabolism and compromise cardiac health later in life.

Measurements of basal d-glucose transport through GLUT1 across the intact plasma membrane of isolated segments from single fast- and slow-twitch skeletal muscle fibres of rat.

AIM: To develop a method for direct measurement of the fluorescent d-glucose analogue 2-NBDG transport across the plasma membrane of single skeletal muscle fibres and derive the theoretical framework for determining the kinetic parameters for d-glucose transport under basal conditions. METHODS: A novel method is described for measuring free 2-NBDG transport across plasma membrane of single rat muscle fibres at rest. The 2-NBDG uptake was >90% suppressed by 100 µM cytochalasin B in both fast-twitch and slow-twitch fibres, indicating that the 2-NBDG transport is GLUT-mediated. Fibres were identified as fast-twitch or slow-twitch based on the differential sensitivity of their contractile apparatus to Sr2+ . RESULTS: The time course of 2-NBDG uptake in the presence of 50 µM 2-NBDG follows a one-phase exponential plateau curve and is faster in fast-twitch (rate constant 0.053 ± 0.0024 s-1 ) than in slow-twitch fibres (rate constant 0.031 ± 0.0021 s-1 ). The rate constants were markedly reduced in the presence of 20 mM d-glucose to 0.0082 ± 0.0004 s-1 and 0.0056 ± 0.0002 s-1 in fast-twitch and slow-twitch fibres respectively. 2-NBDG transport was asymmetric, consistent with GLUT1 being the major functional GLUT isoform transporting 2-NBDG in muscle fibres at rest. The parameters describing the transport kinetics for both 2-NBDG and d-glucose (dissociation constants, Michaelis-Menten constants, maximal rates of uptake and outflow) were calculated from the measurements made with 2-NBDG. CONCLUSION: Free 2-NBDG and d-glucose transport across the plasma membrane of single rat muscle fibres at rest is fast, conclusively showing that the rate-limiting step in d-glucose uptake in skeletal muscle is not necessarily the GLUT-mediated transport of d-glucose.

Effects of intrauterine growth restriction on Ca2+-activated force and contractile protein expression in the mesenteric artery of 1-year-old Wistar-Kyoto rats.

Intrauterine growth restriction (IUGR) affects vascular reactivity in older rats, but at present the causative factors for this change are unknown. Therefore, we investigated downstream events associated with vascular reactivity, specifically, Ca2+-regulated force production and shifts in contractile protein content. The mesenteric artery from male and female 1-year-old Wistar-Kyoto rats was examined using two distinct experimental growth restriction models. Uterine ligation surgery restriction or a sham surgery was conducted at day 18 of pregnancy, whilst a food restriction diet (40% control diet) began on gestational day 15. Extracellular vascular reactivity was studied using intact mesenteric arteries, which were subsequently chemically permeabilized using 50 µM ß-escin to examine Ca2+-activated force. Peak contractile responses to a K+-induced depolarization and phenylephrine were significantly elevated due to an increase in maximum Ca2+-activated force in the male surgery restricted group. No changes in contractile forces were reported between female experimental groups. Sections of mesenteric artery were examined using western blotting, revealing IUGR increased the relative abundance of the voltage-gated Ca2+ channel, inositol-1,4,5-trisphosphate receptor and myosin light chain kinase, in both male growth restricted groups, whereas no changes were seen in females. These findings demonstrate for the first time in 1-year-old rats that changes in vascular reactivity due to IUGR are caused by a change in Ca2+-activated force and shifts in important contractile protein content. These changes affect the Wistar-Kyoto rat in a sex-specific and maternal insult-dependent manner.

The effect of intrauterine growth restriction on Ca2+ -activated force and contractile protein expression in the mesenteric artery of adult (6-month-old) male and female Wistar-Kyoto rats.

Intrauterine growth restriction (IUGR) is known to alter vascular smooth muscle reactivity, but it is currently unknown whether these changes are driven by downstream events that lead to force development, specifically, Ca2+ -regulated activation of the contractile apparatus or a shift in contractile protein content. This study investigated the effects of IUGR on Ca2+ -activated force production, contractile protein expression, and a potential phenotypic switch in the resistance mesenteric artery of both male and female Wistar-Kyoto (WKY) rats following two different growth restriction models. Pregnant female WKY rats were randomly assigned to either a control (C; N = 9) or food restriction diet (FR; 40% of control; N = 11) at gestational day-15 or underwent a bilateral uterine vessel ligation surgery restriction (SR; N = 10) or a sham surgery control model (SC; N = 12) on day-18 of gestation. At 6-months of age, vascular responsiveness of intact mesenteric arteries was studied, before chemically permeabilization using 50 µmol/L ß-escin to investigate Ca2+ -activated force. Peak responsiveness to a K+ -induced depolarization was decreased (P ≤ 0.05) due to a reduction in maximum Ca2+ -activated force (P ≤ 0.05) in both male growth restricted experimental groups. Vascular responsiveness was unchanged between female experimental groups. Segments of mesenteric artery were analyzed using Western blotting revealed IUGR reduced the relative abundance of important receptor and contractile proteins in male growth restricted rats (P ≤ 0.05), suggesting a potential phenotypic switch, whilst no changes were observed in females. Results from this study suggest that IUGR alters the mesenteric artery reactivity due to a decrease in maximum Ca2+ -activated force, and likely contributed to by a reduction in contractile protein and receptor/channel content in 6-month-old male rats, while female WKY rats appear to be protected.

The effects of Suramin on Ca2+ activated force and sarcoplasmic reticulum Ca2+ release in skinned fast-twitch skeletal muscle fibers of the rat.

Suramin has long been used in the treatment of various human diseases. Intravenous infusions of Suramin are commonly administered to patients over extended periods of time but there are a number of significant contraindications with peripheral muscle weakness being one of the most frequently reported. Previous work has shown that even after a single infusion (300 mg kg-1) Suramin remains in skeletal muscle in effective concentrations (11.6 µg mL-1; 84 days) for prolonged periods. These observations provide a strong rationale for investigation of the specific effects of Suramin on skeletal muscle function. Single mechanically skinned fibers were directly exposed to Suramin (10, 100 or 500 µmol L-1) for defined durations (2-10 min) in controlled physiological solutions that mimic the intracellular ionic environment of a fiber. Suramin treatment (10-500 µmol L-1) directly affected the contractile apparatus in a dose-dependent manner causing a decrease in Ca2+-sensitivity (pCa50 = -log (Ca2+) concentration, where 50% of maximum Ca2+- activated force is produced) by 0.14 to 0.42 pCa units and reduction in maximum Ca2+-activated force by 14 to 62%. Suramin treatment (100 µmol L-1 for 10 min and 500 µmol L-1 for 2 min) also caused development of a Ca2+-independent force corresponding to 2.89 ± 4.33 and 16.77 ± 7.50% of pretreatment maximum Ca2+-activated force, respectively. Suramin treatment (100 µmol L-1, 2 min) also increased the rate of sarcoplasmic reticulum (SR) Ca2+ release without significant changes in SR Ca2+ uptake. We report new functional effects for Suramin related to alterations in both the contractile apparatus and SR Ca2+-handling of skeletal muscle that may contribute to the peripheral muscle weakness noted in human pharmacological treatments.

Maternal Nutrient Restriction Alters Ca2+ Handling Properties and Contractile Function of Isolated Left Ventricle Bundles in Male But Not Female Juvenile Rats.

Intrauterine growth restriction (IUGR), defined as a birth weight below the 10th centile, may be caused by maternal undernutrition, with evidence that IUGR offspring have an increased risk of cardiovascular disease (CVD) in adulthood. Calcium ions (Ca2+) are an integral messenger for several steps associated with excitation-contraction coupling (ECC); the cascade of events from the initiation of an action potential at the surface membrane, to contraction of the cardiomyocyte. Any changes in Ca2+ storage and release from the sarcoplasmic reticulum (SR), or sensitivity of the contractile apparatus to Ca2+ may underlie the mechanism linking IUGR to an increased risk of CVD. This study aimed to explore the effects of maternal nutrient restriction on cardiac function, including Ca2+ handling by the SR and force development by the contractile apparatus. Juvenile Long Evans hooded rats born to Control (C) and nutrient restricted (NR) dams were anaesthetized for collection of the heart at 10-12 weeks of age. Left ventricular bundles from male NR offspring displayed increased maximum Ca2+-activated force, and decreased protein content of troponin I (cTnI) compared to C males. Furthermore, male NR offspring showed a reduction in rate of rise of the caffeine-induced Ca2+ force response and a decrease in the protein content of ryanodine receptor (RYR2). These physiological and biochemical findings observed in males were not evident in female offspring. These findings illustrate a sex-specific effect of maternal NR on cardiac development, and also highlight a possible mechanism for the development of hypertension and hypertrophy in male NR offspring.

Sequential effects of GSNO and H2O2 on the Ca2+ sensitivity of the contractile apparatus of fast- and slow-twitch skeletal muscle fibers from the rat.

Reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)) and nitric oxide (NO), have been shown to differentially alter the Ca(2+) sensitivity of the contractile apparatus of fast-twitch skeletal muscle, leading to the proposal that normal muscle function is controlled by perturbations in the amounts of these two groups of molecules (28). However, no previous studies have examined whether these opposing actions are retained when the contractile apparatus is subjected to both molecule types. Using mechanically skinned fast- and slow-twitch skeletal muscle fibers of the rat, we compared the effects of sequential addition of nitrosoglutathione (GSNO), a NO donor, and H(2)O(2) on the Ca(2+) sensitivity of the contractile apparatus. As expected from previous reports in fast-twitch fibers, when added separately, GSNO (1 mM) reduced the Ca(2+) sensitivity of the contractile apparatus, whereas H(2)O(2) (10 mM; added during contractions) increased the Ca(2+) sensitivity of the contractile apparatus. When added sequentially to the same fiber, such that the oxidation by one molecule (e.g., GSNO) preceded the oxidation by the other (e.g., H(2)O(2)), and vice versa, the individual effects of both molecules on the Ca(2+) sensitivity were retained. Interestingly, neither molecule had any effect on the Ca(2+) sensitivity of slow-twitch skeletal muscle. The data show that H(2)O(2) and GSNO retain the capacity to independently affect the contractile apparatus to modulate force. Furthermore, the absence of effects in slow-twitch muscle may further explain why this fiber type is relatively insensitive to fatigue.

Comparison of the effects of inorganic phosphate on caffeine-induced Ca2+ release in fast- and slow-twitch mammalian skeletal muscle.

We compared the effects of 50 mM P(i) on caffeine-induced Ca(2+) release in mechanically skinned fast-twitch (FT) and slow-twitch (ST) skeletal muscle fibers of the rat. The time integral (area) of the caffeine response was reduced by approximately 57% (FT) and approximately 27% (ST) after 30 s of exposure to 50 mM P(i) in either the presence or absence of creatine phosphate (to buffer ADP). Differences in the sarcoplasmic reticulum (SR) Ca(2+) content between FT and ST fibers [ approximately 40% vs. 100% SR Ca(2+) content (pCa 6.7), respectively] did not contribute to the different effects of P(i) observed; underloading the SR of ST fibers so that the SR Ca(2+) content approximated that of FT fibers resulted in an even smaller ( approximately 21%), but not significant, reduction in caffeine-induced Ca(2+) release by P(i). These observed differences between FT and ST fibers could arise from fiber-type differences in the ability of the SR to accumulate Ca(2+)-P(i) precipitate. To test this, fibers were Ca(2+) loaded in the presence of 50 mM P(i). In FT fibers, the maximum SR Ca(2+) content (pCa 6.7) was subsequently increased by up to 13 times of that achieved when loading for 2 min in the absence of P(i). In ST fibers, the SR Ca(2+) content was only doubled. These data show that Ca(2+) release in ST fibers was less affected by P(i) than FT fibers, and this may be due to a reduced capacity of ST SR to accumulate Ca(2+)-P(i) precipitate. This may account, in part, for the fatigue-resistant nature of ST fibers.